1. Field of the Invention
A wide variety of techniques for determining low molecular weight ligands employing conjugates of low molecular weight ligands to fluorescers. In a somewhat more limited number of techniques, the ligand-fluorescer conjugate is joined to a polysaccharide carrier. Many of the ligands and fluorescers are highly hydrophobic. For example, polyiodothyronines conjugated to a fluorescer, such as fluorescein or fluorescein derivatives, can bind non-specifically to a wide variety of proteins which may be encountered in blood or other physiological sample. The problem is alleviated somewhat when the conjugate is bound to a polysaccharide carrier.
Where a polyiodothyronine is conjugated to a fluorescer, the presence of the heavy iodine atom may result in substantial quenching of the fluorescer. Non-specific binding of a protein to the polyiodothyronine may result in a change in the fluorescence. Due to patient sample variation, the degree to which the observed fluorescence changes at constant analyte concentration will vary with the source of the serum. Therefore, it is necessary to find some means to minimize the non-specific effect of the serum proteins on the observed results.
2. Description of the Prior Art
Co-pending application Ser. No. 411,180, filed Aug. 25, 1982, a continuation application of Ser. No. 17,874, teaches the use of a polysaccharide carrier with a polyiodothyronine-fluorescer conjugate to reduce non-specific interference with the fluorescence of the polyiodothyronine-fluorescer conjugate.
U.S. Pat. No. 3,988,943 describes a competitive protein binding assay employing ligand fluorescer conjugates, where the binding of antiligand inhibits the binding of antifluorescer. U.S. Pat. No. 3,996,345 describes an immunoassay employing a chromophore pair, where the chromophores are related by one of the chromophores quenching the fluorescence of the other one of the chromophores, where the amount of quencher brought within quenching distance of the fluorescing chromophore is related to the amount of analyte in the sample. Robbins, "Thyroxine-binding Proteins", Trace Components of Plasma: Isolation and Clinical Significance, Alan, R. Liss, Inc., New York, page 331 (1976) postulated that the inability of prealbumin to bind thyroxine-agarose affinity gels was related to the inability of the thyroxine to orient properly in the protein binding site.